Tetsuo Toraya

Molecular basis for specificities of reactivating factors for adenosylcobalamin‐dependent diol and glycerol dehydratases

Adenosylcobalamin‐dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism‐based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP‐dependent exchange of an enzyme‐bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a)u2003ADP‐dependent cobalamin release and (b)u2003ATP‐dependent dissociation of the resulting apoenzyme–reactivating factor complexes. The inu2003vitro experiments with purified proteins indicated that diol dehydratase‐reactivating factor (DDR)u2003cross‐reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase‐reactivating factor (GDR)u2003did not cross‐reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities inu2003vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase‐bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase‐bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase–DDR complex was cross‐formed, although the diol dehydratase–GDR complex was not formed. There were no specificities of reactivating factors in the ATP‐dependent dissociation of enzyme–reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross‐forms a complex with glycerol dehydratase, and why GDR does not cross‐form a complex with diol dehydratase.

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K+ was mutated to Ala or another amino acid residue. The Eα170A and Dα335A mutants were totally inactive, and the Hα143A mutant showed only a trace of activity, indicating that Glu-α170, Asp-α335, and His-α143 are catalytic residues. The Qα141A, Qα296A, and Sα362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-α296 is important for substrate binding and Gln-α296 and Gln-α141 for preventing the enzyme from mechanism-based inactivation. The Eα221A, Eα170H, and Dα335A did not form the (αβγ)2 complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-α170 and Asp-α335 mutants, Eα170D and Eα170Q were 2.2 ± 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dα335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue α170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-α170 and Asp-α335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-α170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.

Mechanism-based Inactivation of Coenzyme B12-dependent Diol Dehydratase by 3-Unsaturated 1,2-Diols and Thioglycerol

The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.

Diol dehydratase‐reactivating factor is a reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase

Adenosylcobalamin‐dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co–C bond. The damaged cofactor remains tightly bound to the active site. The DD‐reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+. Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1u2003:u20031 and 1u2003:u20032 complexes with the reactivase in the presence of ADP and Mg2+. Upon complex formation, one βu2003subunit was released from the (αβ)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD βu2003subunit and the reactivase βu2003subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

cDNA cloning, expression, and characterization of methyl-CpG-binding domain type 2/3 proteins from starfish and sea urchin.

Two kinds of cDNAs that are highly homologous to mammalian MBD2 and MBD3 cDNAs were cloned from ovary of the starfish Asterina pectinifera. They are splicing variants and designated sMBD2/3a and sMBD2/3b cDNAs. sMBD2/3a cDNA spans 1378 bp and consists of a 48-bp upstream untranslated region, a 807-bp open reading frame encoding sMBD2/3a, and a 523-bp downstream untranslated region. sMBD2/3a and sMBD2/3b cDNAs encode proteins with predicted molecular weights of 30,724 and 29,635 consisting of 268 and 260 amino acid residues, respectively. The deduced amino acid sequences of these two are identical from residues 1 to 255, but different from residues 256 to the C-terminal ends. sMBD2/3a is expressed in all the tissues of starfish, whereas sMBD2/3b is highly expressed in ovary and oocytes, slightly in testis, but not in somatic cells. As suggested from the whole-genome sequence of Strongylocentrotus purpuratus, a sea urchin MBD2/3 cDNA was cloned from eggs of Hemicentrotus pulcherrimus and designated suMBD2/3 cDNA. It encodes a protein with predicted molecular weight of 30,778 consisting of 274 amino acid residues. All the three echinodermal MBD2/3 proteins consist of a methy-CpG-binding domain (MBD) and a coiled-coil domain, and only sMBD2/3a contains a glutamate-rich C-terminal region, a key mark in vertebrate MBD3. The three MBD2/3 proteins expressed in Escherichia coli and purified to homogeneity were capable to bind specifically to methylated DNA. It was shown that sMBD2/3a exists as dimer or in the monomer-dimer equilibrium, whereas sMBD2/3b and suMBD2/3 exist as monomer and dimer, respectively.

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase

[ω‐(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5′, which is critical for Co‐C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia‐lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia‐lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia‐lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co‐C bond cleavage upon binding to apoenzymes.

Essential Roles of Nucleotide-Switch and Metal-Coordinating Residues for Chaperone Function of Diol Dehydratase-Reactivase

Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eβ31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R β subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R β and DD β subunits, Gluβ97 of DD would coordinate to Mg(2+). The complex of Eβ97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluβ97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.

Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms.

Adenosylcobalamin (coenzyme B12) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg2+ by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P2(1)2(1)2(1) and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 A, and diffract to 2.0 A. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P2(1)2(1)2(1) and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 A and diffract to 3.0 A. Crystals of both forms were suitable for structural analysis.

Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2 with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO(-) group at residue α287 was absolutely required for activity and coenzyme Co-C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co-C bond homolysis, stabilizes the transition state for the migration of NH2 from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2 migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+). The activity was very sensitive to the position of -COO(-) at α362. Aspα362 would assist Co-C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+) and electrostatic interaction.

Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis

The X‐ray structure of the diol dehydratase–adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine‐binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6‐NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε‐NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19–2% as active as the wild‐type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58–76% of the wild‐type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme–cobalamin interaction. The Sα224A and Sα224N mutants showed a kcat/kinact value that was less than 2% that of the wild‐type, whereas for Lysβ135 mutants this value was in the range 25–75%, except for the Kβ135E mutant (7%). Unlike the wild‐type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt–carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′‐Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism‐based one.